Protein phosphorylation is a common natural post-translational modification regulated by kinases and phosphatases and critical for cellular signaling networks. Studying phosphoproteins and their associated signaling networks is often difficult due to challenges in phosphoprotein expression and isolation. Kinase deoxyribozymes will be developed for the site-specific phosphorylation of proteins. Multiple in vitro selection approaches will be used to identify small peptide-sequence-specific and motif-specific kinase deoxyribozymes. Parallel efforts will pursue the phosphorylation of large, full-length proteins using aptamer-linked deoxyribozymes. This modular approach will allow the aptamer to serve as the protein-binding domain while the kinase deoxyribozyme is the catalytic domain, thereby leading to phosphorylation of the protein. Protein phosphorylation site specificity will be achieved in several ways: by adjusting the linker between the aptamer and deoxyribozyme, by using aptamers that bind to different regions of the protein, and by using peptide-sequence-specific deoxyribozymes. Comparison of these approaches will allow us to learn the best way to site-specifically phosphorylate large protein substrates using DNA catalysts. Biologically important site-specifically modified phosphoproteins as generated by kinase deoxyribozymes will be studied to increase our knowledge of the role of phosphoproteins in nature.